Cells interact with biochemically and structurally forms of extracellular matrix in different tissues, at different stages of embryonic development, and during adult wound repair. This project focuses on addressing the following major questions concerning the mechanisms of these cell-extracellular matrix interactions: 1. What unique mechanisms do mammalian cells use to migrate through different three-dimensional (3D) extracellular matrix environments compared to flat cell culture substrates? 2. What distinct signal transduction mechanisms control cell behavior in different 3D microenvironments? We are exploring which elements of the classical models of cell motility and signaling established using regular 2D cell culture are valid in the structurally complex 3D environments found in tissues. We have identified a variety of differences in cell migration and signaling between 2D and 3D environments, but also between different 3D environments, such as collagen-based matrices that differ in architecture compared to fibronectin-rich cell-derived matrices. In these matrices, primary human dermal fibroblasts migrate at different speeds and with distinct modes of migration, with one major difference being the use of actin polymerization-based lamellipodial migration in various collagen-based matrices versus intracellular pressure-based lobopodial migration in linearly elastic cell-derived matrix. Human fibroblastic cells in 3D collagen environments, regardless of their local stiffness and micro-architecture, were previously shown to display markedly elevated levels of integrin activation and clustering compared to cells cultured on flat collagen matrices in cell culture. This enhanced integrin activation was accompanied by a requirement for cellular contractility, apparently in order for these cells to be able to detach effectively from their enhanced integrin-mediated attachments to 3D fibrils to permit efficient cell migration. The next phase of this study is characterizing the myosin II-dependent mechanisms of human fibroblast migration in 3D collagen microenvironments, whereas myosin II is not required when these same cells migrate on 2D surfaces. In addition, we are comparing the alterations in mechanisms after malignant transformation by an in-depth characterization of the modes of 3D cell migration used by human tumor cells. We had previously established that primary human fibroblasts migrating in a regular non-linearly elastic 3D environment such as a collagen matrix, or even in a protease-degraded cell-derived matrix that was also non-linearly elastic, used classical lamellipodial migration. These cells can readily switch to the new mode of lobopodial migration in a linearly elastic 3D cell-derived matrix by using their nucleus as a piston to form high-pressure anterior protrusions that effectively extend the cell forward. Because tumor cells often locally degrade their adjacent 3D matrix environment, we evaluated whether proteolysis might affect the mode of tumor cell 3D migration. We found that unlike primary fibroblasts, the nuclear piston is inactive in their malignant counterpart fibrosarcoma cells. Protease inhibition rescued the nuclear piston mechanism in polarized fibrosarcoma cells. and generated the characteristic anterior compartmentalized pressure. Thus, simply inhibiting protease activity during polarized tumor cell 3D migration is sufficient to restore the nuclear piston migration mechanism characteristic of normal human fibroblasts. We recently reviewed the multiple mechanisms that cells can use to migrate in 3D environments. Although there are quite distinct modes of cell migration, they often rely on the same intracellular components, such as cellular actomyosin, for successful cell migration. Key elements identified to date include the ratio of actomyosin contractility to sell-matrix adhesion and the extent of confinement of the nucleus by a 3D microenvironment, which requires contractility to squeeze the nucleus through matrix pores. Consequently, the nucleus of a cell can both determine the ease of migration through matrix pores according to its size and stiffness, as well as serving as an active component when used as a nuclear piston.